1、Designation: E2839 11Standard Test Method forProduction of Clostridium difficile Spores for Use inEfficacy Evaluation of Antimicrobial Agents1This standard is issued under the fixed designation E2839; the number immediately following the designation indicates the year oforiginal adoption or, in the
2、case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONSporulation in Clostridium diffcile is not as rapid or as efficient as in other species
3、and it is generallydifficult to produce C. diffcile spores of high titer in the laboratory (1, 2).2Although quantitative testmethods are available for testing sporicidal products, a standardized method for generating sporesuspensions of C. diffcile of high titer (8 log10/mL) and purity ($95 % spores
4、) is not available andwould be necessary in order to conduct performance testing required for registration purposes (3). Thespore suspensions resulting from practice of this test method are appropriate for use in accepted testmethods for measuring the sporicidal efficacy of antimicrobial formulation
5、s (4).1. Scope1.1 This test method is for producing C. diffcile spores toevaluate antimicrobial formulations for their sporicidal activ-ity.1.2 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLP) are required and tofollow them when appropriate.1.3 This st
6、andard may involve hazardous materials, chemi-cals, and microorganisms and should be performed only bypersons with formal training in microbiology.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not pu
7、rport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Terminology2.1 Definitions:2.1.1 CFU, adj
8、/ncolony-forming units; the number ofspores or microorganisms that can form colonies (clusters ofmicroorganisms visibly growing on the surface of a solid agarmedium) in spread plates, as an indication of the total numberof viable spores/microorganisms in a sample.2.1.2 QC, adj/nquality control (QC)
9、is the application ofprocedures, products, or services to meet a laboratorys speci-fied standards of quality.2.1.3 pre-reduced medium, adj/nan agar or broth manu-factured and sterilized in an oxygen-free environment, andpackaged individually in air-tight sealed pouches or bags.2.1.4 density gradient
10、 medium, adj/nHistoDenz (trade-marked)3is a non-ionic gradient medium used here to separatespores from vegetative cells and cell fragments on the basis ofdensity.2.1.5 purified spores, adj/nwhen spore concentrationreaches $95 % as vegetative cells and cell fragments areseparated by the density gradi
11、ent medium.2.1.6 toxigenic strain, adj/npossesses either toxin A gene(tcdA+) or toxin B gene (tcdB+) or both.3. Summary of Test Method3.1 This test method provides detailed instructions for theculture, maintenance and sporulation of C. diffcile on a specificagar medium incubated in an anaerobic envi
12、ronment for 7 to 10days. Monitoring is performed by phase-contrast microscopyto ensure sporulation is underway and to determine when thespore concentration reaches $90 %, the optimal time ofharvest. Upon harvesting, spores are washed several times withsaline-Tween 80, treated with heat to inactivate
13、 any remainingviable vegetative cells, and purified using a density gradientmedium to remove inactivated vegetative cells and cell frag-ments, with a target spore-purity of$95 %. Purified spores are1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Al
14、ternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Aug. 1, 2011. Published September 2011. DOI:10.1520/E2839-11.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3The sole source
15、 of supply of HistoDenz (trademark) (Cat. No. D2158) knownto the committee at this time is Sigma-Aldrich, St. Louis, MO. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of there
16、sponsible technical committee,1which you may attend.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.enumerated on specific agar-based recovery medium for titerdetermination and assessed for quality using a quantitativeacid-resistance
17、 test.4. Significance and Use4.1 This test method describes a procedure for preparing aspore suspension of C. diffcile strain ATCC 43598 that meetsspecific criteria necessary for efficacy testing of antimicrobialsdesigned to eliminate C. diffcile contamination from environ-mental surfaces. The accep
18、tability criteria for the spore sus-pension are: (1) a viability titer of 8 log10/mL, (2) purity of$95 %, and (3) that spores be resistant to 10 min of exposureto 2.5 M HCl.5. Apparatus5.1 Biosafety cabinet (BSC, Type B2, Class II)Recommended for maintaining an aseptic work environment.5.2 Sterile c
19、entrifuge tubesPolypropylene, 15 mL and 50mL graduated plastic centrifuge tubes with conical bottoms.5.3 Centrifuge with swinging-bucket rotorTo allow sedi-mentation of spores for washing and/or concentration.5.4 MicropipetteCalibrated.5.5 Positive displacement pipetteTo inoculate steel carri-ers wi
20、th spores.5.6 TimerAny certified timer that can display time inseconds.5.7 Test tubesReusable or disposable 20 3 150 mm forcultures/subcultures.5.8 Inoculating loop10 L transfer loop.5.9 Anaerobic chamberSupported by a gas mixture con-sisting of 10 % hydrogen, 5 % CO2, and 85 % N2. Alterna-tively, a
21、n activated anaerobic jar can be used according tomanufacturers instructions for ensuring an anaerobic environ-ment.5.10 Anaerobic incubatorUse an incubator at 36 6 1Cplaced inside the anaerobic chamber to support the growth ofthe organism. Alternatively, use an activated anaerobic jarcontaining ino
22、culated plates that is placed inside an aerobicincubator at 36 6 1C. Plates must be incubated in ananaerobic environment at 36 6 1C for growth to occur.5.11 Microscope with 103 eyepiece and 403 and 1003(oil) objectives with phase contrast option.5.12 Vortex mixer.5.13 Serological pipettesSterile sin
23、gle-use pipettes of10.0, 5.0, 1.0 mL capacity.5.14 Cell ScraperTo gently scrape plates to removespores for harvesting.5.15 Plate spreaderTo spread inocula on agar to create auniform lawn.5.16 Microcentrifuge tubesSterile 1.5-mL low-retention(siliconized) microcentrifuge tubes.5.17 CryovialsSterile 2
24、.0 mL cryovials.5.18 Parafilm.6. Media and Reagents6.1 Culture Media:6.1.1 Reinforced clostridial medium (RCM)For use inrehydrating lyophilized/frozen vegetative culture of test organ-ism. Prepare RCM according to manufacturers instructions,and pre-reduce in an anaerobic environment for 24 6 2 h pri
25、orto use.6.1.2 RCM plus 15 % glycerol (Cryoprotectant)For use asmaintenance and cryopreservation medium for vegetative fro-zen stock (VFS) cultures. Prepare RCM and add 15 % glycerol,autoclave for 20 min at 121C, and pre-reduce (6.1.1).6.1.3 Sporulation mediumCDC anaerobic 5 % sheepblood agar (CABA)
26、, commercially available pre-reduced.46.1.4 Recovery media for enumeration of viable sporesPre-reduced brain-heart infusion agar with yeast extract, horseblood and sodium taurocholate (BHIY-HT).46.2 Phosphate-buffered saline (PBS)Prepare 103 stocksolution of PBS by dissolving 492 g PBS powder in 5 L
27、 ofdeionized water. Dilute 1:10 (1 part 103 solution plus 9 partsdeionized water) to obtain 13 solution, distribute into bottlesand autoclave for 20 min at 121C.6.3 Phosphate-buffered saline (PBS) containing 0.1 %Tween 80 (ST80)Washing reagent; add 2.0 mL of polysor-bate 80 (Tween 80, or equivalent)
28、 to 2.0 L of PBS (13)solution ina2Lvolumetric flask and bring solution to volumewith PBS. Distribute into bottles and autoclave for 20 min at121C.6.4 WaterSterile deionized water (5).6.5 Hydrochloric acidPrepare 2.5 M HCl from 5 M HCl.6.6 HistoDenzPrepare a 50 % (w/v) solution in deionizedwater. Thi
29、s is a density gradient medium. Pass the solutionthrough a sterile 0.45 m filter.7. Test Organism7.1 Clostridium diffcile (ATCC 43598), a toxigenic strain(tcdA-, tcdB+), can be obtained from a reputable vendor. Thestrain produces Toxin B only (presence of tcdB gene by PCR).The organism is a Gram-pos
30、itive, strictly anaerobic, spore-forming bacterium that produces flat, gray, and irregularcolonies on the surface of CABA medium within 48 h at 3661C.8. Hazards8.1 The test organism (C. diffcile, ATCC 43598) must beincubated under strict anaerobic conditions and in accordancewith local biosafety pra
31、ctices or those recommended by theU.S. Centers for Disease Control and Prevention/NationalInstitutes of Health (CDC/NIH) for organisms at BiosafetyLevel II (6). Processing of spores can be conducted in anaerobic environment (for example, inside a BSC); all incuba-tion for growth, however, must be pe
32、rformed anaerobically.8.2 Use suitable personal protective equipment (PPE) andother appropriate safety devices when handling hydrochloricacid and other hazardous chemicals. Consult relevant MaterialSafety Data Sheets (MSDS) in advance for specific details onsafe manipulation of such chemicals and co
33、rrective action incase of spills or exposure.4The sole source of supply of the CABA (Cat. No. AS-646) and BHIY-HT (Cat.No. AS-6463) known to the committee at this time is Anaerobe Systems, MorganHill, CA. If you are aware of alternative suppliers, please provide this informationto ASTM International
34、 Headquarters. Your comments will receive careful consid-eration at a meeting of the responsible technical committee,1which you may attend.E2839 1129. Preparation of Frozen Stock Cultures of TestOrganism9.1 Preparation of Inoculum:9.1.1 Clostridium diffcile received in lyophilized vegetativeform:9.1
35、.1.1 Reconstitute contents of the lyophilized culture with0.5 mL of sterile pre-reduced RCM in an anaerobic environ-ment according to manufacturers instructions.9.1.1.2 After rehydration, aseptically transfer the vial con-tents to a tube containing 4 6 1 mL of pre-reduced RCM, andmix by gentle vorte
36、xing.9.1.2 Clostridium diffcile received as frozen vegetativeculture:9.1.2.1 Thaw frozen culture at room temperature.9.1.2.2 Transfer the contents to a tube containing 4 6 1mLof sterile pre-reduced RCM in an anaerobic environment, andmix by gentle vortexing.9.2 Inoculation of CABA Plates for Vegetat
37、ive Stock Cul-ture:9.2.1 Inoculate by spread-plating each of five CABA plates(100-cm diameter) with 100 L of the reconstituted/dilutedculture of C. diffcile.9.2.2 Streak one CABA plate for isolation to check forculture purity.9.2.3 Invert plates and incubate anaerobically at 36 6 1Cfor 48 6 4h.9.3 H
38、arvest of CABA Plates for Stock Culture:9.3.1 Following incubation (9.2.3), add approximately 2 mLof sterile and pre-reduced cryoprotectant (6.1.2) to each CABAplate.9.3.2 Using a sterile cell scraper, gently scrape culture fromthe surface of one plate, aspirate with a pipette and transfer toa 15-mL
39、 conical tube. Repeat this process for the remainingplates.9.3.3 Pool the cryoprotectant suspensions, mix thoroughly,and pipette 1 to 1.5 mL aliquots into cryovials; cap tightly.9.3.4 Store the cryovials at #70C. These tubes are theFrozen Stock Culture (FSC).9.4 Evaluation of Viable Titer of FSC:9.4
40、.1 Approximately 7 6 1 days after freezing, thaw a stockculture cryovial at room temperature inside an anaerobicchamber.9.4.2 Vortex suspension thoroughly, and dilute 1 mL in a1:10 series out to 106in ST80 (6.3).9.4.3 Spread-plate 100 L of diluted suspension onBHIY-HT in duplicate.9.4.4 Invert plate
41、s and incubate anaerobically at 36 6 1Cfor 48 6 4 h. Record the number of CFU/plate to determine theviable titer/mL, which should be 8 log10/mL to ensure thatFSC contains a sufficiently high titer to withstand long-termstorage at #70C.10. Preparation of a Test Spore Suspension from FSC10.1 Inoculati
42、on of CABA Plates:10.1.1 As a part of QC, streak three CABA plates with afrozen stock culture of test organism. Incubate two platesanaerobically, and the third one aerobically at 36 6 1C for48 6 4 s. Do not use the culture if there is any growth on theplate incubated aerobically. Inspect plates incu
43、bated anaerobi-cally for purity and colony characteristics typical of C. diffcile.10.1.2 Inoculate 10 mL of pre-reduced RCM with anisolated colony from a CABA plate and mix well by vortexing.Incubate anaerobically at 36 6 1C for 24 6 2h.10.1.3 After incubation, inoculate each of a minimum of tenCABA
44、 plates with 100 L of the RCM broth culture. Spreadthe inoculum evenly using a disposable sterile spreader tocreate a lawn.10.1.4 Seal culture plates with Parafilm, or equivalent, toprevent dehydration during the extended anaerobic incubation.Invert plates and incubate anaerobically for 7 to 10 days
45、 at36 6 lC and $70 % relative humidity. Maintenance of rela-tive humidity is not required if an anaerobic jar is used.10.1.5 Open one or two plates after about 24 h of incubationto inspect for confluent growth. Do not continue with theincubation if growth is not confluent. Wet-mount samples of C.dif
46、fcile from the plates periodically during the first 2 to 6 daysof incubation, and daily on days 7 to 10, for inspection underphase-contrast microscopy. Note degree of conversion ofvegetative cells to spores and estimate the approximate ratio ofspores to vegetative cells to determine the optimal time
47、 forharvesting. Under phase-contrast, spores appear bright andovular, while vegetative cells appear dark and rod-shaped.10.2 Harvesting CABA Plates Inside a BSC (that is, aerobicenvironment):10.2.1 When the percent of spores reaches $90 %, discon-tinue incubation in anaerobic environment and remove
48、theCABA plates into a BSC. Harvest growth from each plate byadding approximately 5 mL of ST80 to each plate, and gentlyscrape the surface of the plate with a cell scraper to dislodgethe spores. Do not break the surface of the agar, and avoidcollecting agar fragments, insofar as possible.10.2.2 Using
49、 a 10 mL sterile serological pipette, aspirate asmuch of the microbial suspension as possible from each plate,and pool it in sterile 50-mL plastic conical tubes. Cap the tubestightly for centrifugation. For proper balancing, there must beat least two 50-mL plastic tubes of the same size with the samevolume, and pairs of tubes must be positioned in bucketsdiametrically opposite one another.10.3 Washing the Spore Suspension by Centrifugation:10.3.1 Centrifuge tubes at 4500 3 g for 15 min.10.3.2 Discard the supernatant a
